Breast cancer is the most common malignancy in women worldwide. While advance have been made in treatment this disease still accounts for the second largest number of cancer deaths in women in the US. These deaths are largely due to metastatic disease. This proposal is based upon observations by project leaders (PL) and other members of the PPG that tumor associated macrophages (TAMs) play a critical role in promoting the rate-limiting steps of metastasis - tumor cell intravasation and extravasation. Central to these observations was the definition by the PL of TAM sub-populations defined by their transcriptome and cell surface markers. Thus different populations have individual functions including in the primary tumor the stimulation of angiogenesis, tumor cell invasion and intravasation as well as at metastatic sites extravasation and persistent growth. In this proposal we have three specific aims: Aim 1: Mechanisms for Monocyte Recruitment and Macrophage Differentiation in the Primary Tumor. In this aim the mechanism of recruitment and differentiation of the different populations to the primary tumor will be studied with a particular focus upon those macrophages that stimulate invasion and intravasation. We will use novel methods for fate mapping macrophages to explore their origin and plasticity in the tumor. Aim 2: Molecular basis for the Promotion of Extravasation by of Metastasis Associated Macrophages. This aim will define the signaling pathways that convert macrophages into those that promote extravasation of tumor cells. It will discover the mechanism whereby macrophages enable tumor cells to egress through the endothelial layer. Aim3: Definition of Pro-Tumoral Functions of Human breast Cancer TAMs Studies will be translated from mouse models into human breast cancer. Human TAMs from various disease sub-types will be isolated followed by ex vivo assays for function. These assays together with transcriptional profiles will define signaling pathways and determine whether they parallel those found in mice. Together these studies will enable therapeutic targets aimed at sub-populations of macrophages rather than the pan- macrophage therapeutics currently in trial that have toxicities because that target all resident macrophages. RELEVANCE (See instructions): In the US -250,000 new cases of invasive ductal breast carcinoma are diagnosed each year with 40,000 deaths. Over the last decade statistics show an improvement in survival for women with local disease but no change for those with metastatic disease. This depressing statistic demands new therapeutic approaches. In this application we propose that one strategy to improve treatment is to target the tumor microenvironment and particularly the macrophages within it in order to remove their pro-tumor support.